MMSET Regulates IRF4, a Transcription Factor Critical for the Survival of T(4;14) Myeloma Myeloma Cells, and Its Silence Potentiates the Effect of Antimyeloma Agents

Multiple myeloma (MM) is a genetically complex disease that is becoming more common in today’s ageing population. Approximately 50% of MM harbour recurrent translocations involving the immunoglobulin heavy chain (IgH) locus on chromosome 14q32. The translocation t(4;14)(p16;q32) is one of the most common translocation in MM and is associated with very poor prognosis. The multiple myeloma SET domain (MMSET), involved in the fusion to the IgH locus in t(4;14) MM, is universally overexpressed and has been suggested to play an important role in tumorigenicity in t(4;14) MM. In order to identify downstream functional targets of MMSET, we knocked down MMSET expression with shRNAs in KMS11, a t(4;14) MM cell line, and identified differentially expressed genes by gene expression microarray analysis. The results identified 321 down-regulated and 375 up-regulated genes upon MMSET knockdown. Of interest, the knockdown reduced the transcription levels of the transcription factor IRF4 (interferon regulatory factor 4), which has been shown recently to be required for myeloma cell survival. Quantitative PCR (QPCR) analysis confirmed the transcription levels of IRF4 were reduced upon MMSET knockdown in t(4;14) MM cells (KMS11, KMS18, KMS28BM). Western-blots analysis indicated MMSET knockdown reduced protein levels of IRF4, c-Myc and PARP, and IRF4 knockdown could also reduce protein levels of c-Myc and PARP. Flow cytometric analysis indicated silencing of MMSET or IRF4 could induce apoptosis in t(4;14) MM cells. QPCR and Western-blot analysis revealed that ectopic expression of MMSET could increase IRF4 expression in KMS11 cells. Using chromatin immunoprecipitation (ChIP), both MMSET and NFκB can bind to the promoter region of IRF4, and the binding region of MMSET is located 1 kb upstream of that of NFκB. Furthermore, endogenous NFκB p65 could be coprecipitated by anti-MMSET antibody, indicating the association of MMSET and NFκB. These data suggest that MMSET may be a coactivator for NFkB in the regulation of IRF4 expression. Luciferase reporter assays in KMS11 cells showed the activity of IRF4 promoters were decreased significantly upon MMSET knockdown, suggesting MMSET is an important functional element for IRF4 promoter. Cell proliferation assays and apoptosis analysis showed that silencing of MMSET could sensitize t(4;14) MM cells to Bortezomib and Melphalan killing. In myeloma xenograft, combined MMSET silencing with Melphalan or Bortezomib resulted in significantly greater inhibition of t(4;14) MM tumorigeness than treatments with either drugs alone. Importantly, we found both of Melphalan and Bortezomib could also reduce protein levels of MMSET and IRF4. QPCR analysis indicated that they inhibited MMSET and IRF4 expression through transcription control. These results might partly explain the additive mechanism of combination of MMSET knockdown and Melphalan or Bortezomib. Overall, our data indicated MMSET is involved in the regulatory network of IRF4 that is critical for t(4:14) MM cell survival, and its silence potentiates the effect of antimyeloma agents in vitro and in vivo.